Peptide combination for the treatment of cancer

a cancer and peptide technology, applied in the direction of peptide sources, animals/human peptides, protease inhibitors, etc., can solve the problems of moderate cytotoxicity and narrow activity spectrum of peptides

Inactive Publication Date: 2003-03-13
DABUR PHARM LTD
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Benefits of technology

0058] Experiments were conducted to study the cytotoxic effect of the combination on 12 human tumor cell lines using the three-day MTT cytotoxic assay. These cell lines were K562 (human leukemia), MOLT-4 (human lymphoma), L132, A549 (human lung carcinoma), MCF-7, HBL100, MDA.MB.453 (human breast), SW620, PTC, CoLo205, HT29, CaCO.2 (human colon), HuTu80 (human duodenum), Hu746T (human stomach), SKO.007 (human myeloma), SK.MEL.28 (human melanoma). Briefly, cells from the 12 human tumor cell lines were incubated in a 96-well culture plate (approximately 50,000 cancer cells in each well) for 72 hours at 37.degree. C. in a CO.sub.2 incubator. The combination SEQ ID NO: 1+SEQ ID NO: 2+SEQ ID NO: 3+SEQ ID NO: 4, all at 10.sup.-8 M concentration (20 ul per well) was added to the wells of all the treated samples at time 0, 24, and 48 hours. The controls were cells from 12 tumor cell lines that were not treated with the combination. At the end of 72 hours, stock MTT solution was added to each well, and incubation continued for one additional hour. After adding SDS-0.01N HCl, the plate was read at 540 nm. The percent cytotoxicity caused by the combination in each of the 12 cell lines is listed in Table-II.
0059] Human colon adenocarcinoma cells cultured to 70% confluence were harvested using 0.05% trypsin-0.2 mM EDTA solution. Subsequently, the cells were re-plated in medium supplemented with 10% FCS, in 96 well micro titer tissue culture plates at a density of 5000 cells/well. The plates were incubated overnight to allow complete reattachment of the cells. The medium was replaced with RPMI-1 640 containing 2.5% FCS. The specific cellular signaling inhibitors for different signaling pathways were added in pre-determined optimal non toxic concentrations (in pM-nM range) once every 24 hours, for a period of 72 hours. The control cells were incubated with the vehicle alone. After 72 hours, the cell viability was quantitated by the MTT assay.
0060] The inhibitors to specific cellular signaling pathways were added individually along with the peptide combination SEQ ID NO: 1+SEQ ID NO: 2+SEQ ID NO: 3+SEQ ID NO: 4, all at 10.sup.-9M concentration once every 24 hours, for a period of 72 hours. The control cells were incubated with the peptide combination or with the vehicle alone. The cell viability was quantitated by the Formazan based MTT assay. The percent abrogation in the cytotoxicity of the peptide combination when co-incubated with the specific cellular signaling inhibitor was calculated.
0061] The treatment of cells with the peptide combinati

Problems solved by technology

However, when used individually, these peptides had a narro

Method used

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  • Peptide combination for the treatment of cancer

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Experimental program
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example 2

The Combination was Prepared in the Following Way

[0055] A stock solution for each of the four peptides (SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO:3 and SEQ ID NO: 4) is prepared with a pH of approximately 3.5 to 7.0 but preferably 4.0 to 5.5. Although sterile phosphate buffered saline was used to prepare each stock solution for the testing described in the following example, other diluents may be used such as buffered saline, isotonic NaCl, Ringer's solution, water, distilled water, polyethylene glycol (neat or in water), Tween in water, dimethylsulfoxide upto 50% in water, propylene glycol (neat or in water), phosphate buffered saline, balanced salt solution, glycerol, and other conventional fluids that are suitable for parentral administration. To obtain a pH in the range of approximately 3.5 to 7.0, for each stock solution, the pH can be adjusted by using 1N HCl for lowering the pH or 1N NaOH for raising the pH, although other buffers such as citrate buffer, phosphate buffer and the l...

example 3

In Vitro Cytotoxicity Studies on the Combination

[0057] The combination SEQ ID NO:1+SEQ ID NO:2+SEQ ID NO:3+SEQ ID NO: 4, all in 10.sup.-8 M concentration was tested for cytotoxicity against 12 human tumor cell lines. It was also tested against primary human colon adenocarcinoma cells and other colon cancer cell lines. Briefly, a one day MTT cytotoxicity assay was performed, which is based on the principle of uptake of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), a tetrazolium salt, by the metabolically active cells where it is metabolized by active mitochondria into a blue colored formazan product that is read spectrophotometrically [Mosmann, 1983]. MTT was dissolved in phosphate buffered saline with a pH of 7.4 to obtain an MTT concentration of 5 mg / ml; the resulting mixture was filtered through a 0.22 micron filter to sterilize and remove a small amount of insoluble residue. For each type of tumor cell, 20,000 to 50,000 cells were seeded in a 96-well cultur...

example 4

Cytotoxicity Abrogation Assays for Identification of Cellular Signaling Pathways Modulated by Peptide Combination

[0059] Human colon adenocarcinoma cells cultured to 70% confluence were harvested using 0.05% trypsin-0.2 mM EDTA solution. Subsequently, the cells were re-plated in medium supplemented with 10% FCS, in 96 well micro titer tissue culture plates at a density of 5000 cells / well. The plates were incubated overnight to allow complete reattachment of the cells. The medium was replaced with RPMI-1 640 containing 2.5% FCS. The specific cellular signaling inhibitors for different signaling pathways were added in pre-determined optimal non toxic concentrations (in pM-nM range) once every 24 hours, for a period of 72 hours. The control cells were incubated with the vehicle alone. After 72 hours, the cell viability was quantitated by the MTT assay.

[0060] The inhibitors to specific cellular signaling pathways were added individually along with the peptide combination SEQ ID NO: 1+SEQ...

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Abstract

The present invention relates to a combination of peptides that may be used for treatment of cancer. The combination of peptides modulates multiple cellular pathways implicated in cell proliferation by altering the levels of key intracellular molecules thereby showing a broad spectrum of anticancer activity. The invention also relates to a pharmaceutical composition containing a combination of such peptide analogs.

Description

[0001] The present invention relates to a combination of peptides that may be used for treatment of cancer. The combination of peptides modulates multiple cellular pathways implicated in cell proliferation by altering the levels of key intracellular molecules thereby showing a broad spectrum of anticancer activity. The invention also relates to a pharmaceutical composition containing a combination of such peptide analogs.[0002] The anticancer drugs currently used for the treatment of adenocarcinomas have a limited spectrum of antitumor activity and a narrow therapeutic index. For the most part, the current choices for the oncologist are among alkylating agents, antimetabolites, DNA binders, tubulin interactive antimitotics, topoisomerase inhibitors, anti-hormones, and a few other agents of mixed or unknown mechanisms. Most of these drugs as a group are similar, not only with respect to the spectrum of clinical antitumor activity and toxicity, but also with respect to their mechanism...

Claims

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Application Information

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IPC IPC(8): A61K38/08A61K38/10A61K38/22A61K38/31
CPCA61K38/10A61K2300/00A61K38/08
Inventor BURMAN, ANAND C.MUKHERJEE, RAMAPRASAD, SUDHANANDJAGGI, MANUSINGH, ANU T.
Owner DABUR PHARM LTD
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